Fluorescent chloramphenicol derivatives for determination of chloramphenicol acetyltransferase activity

ABSTRACT

Fluorescent compounds useful in the determination of chloramphenicol acetyltransferase (CAT) enzyme activity are described. The compounds are fluorescent derivatives related in structure to chloramphenicol comprising a base, ##STR1## substituted at one to five aromatic ring positions by substituents, which may be the same or different, that are alkyl-, hydroxy-, alkoxy-, aryl-, halo-, nitro-, amino-, alkylamido-, or arylamido-; and a fluorescent moiety linked to the base at --NH-- by an aliphatic chain of 1-12 atoms other than hydrogen, where such atoms are carbon, nitrogen, oxygen, or sulfur, or combinations thereof. The substrate compounds are acylated in the presence of CAT to produce fluorescent mono- and diacylated products, which are then physically separated from the reaction mixture and quantitated by means of their fluorescence and/or absorbance. Fluorescent molecules conjugated to chloramphenicol include derivatives of fluorescein, rhodamine, coumarin, dimethylaminonaphthalene sulfonic acid (dansyl), pyrene, anthracene, nitrobenzoxadiazole (NBD), acridine and dipyrrometheneboron difluoride.

This invention was made with government support under grants nos. 5 P51RR00163 and GM 38987 awarded by the National Instituted of Health. Thegovernment has certain rights in this invention.

This is a continuation of application Ser. No. 07/321,494, filed on Mar.9, 1989, now abandoned.

FIELD OF THE INVENTION

This invention describes fluorescent compounds useful in thedetermination of chloramphenicol acetyltransferase (CAT) enzymaticactivity. The measurement of CAT activity is widely used to studytranscriptional control sequences in a number of fields, some of whichinclude endocrinology, microbiology, virology, genetics, oncology,developmental biology and molecular biology.

BACKGROUND OF THE INVENTION

The study of gene expression has required the development of genes whichare relatively easy to assay and can serve as markers to monitorexpression of foreign genetic material which has been introduced intocells. The foreign genomic DNA fragments are inserted into a recombinantplasmid along with a marker gene, and after introduction of the plasmidinto the target cell, the quantity of protein coded for by the markergene is determined. When compared to the quantity of protein produced bya plasmid without the insert, the result is indicative of the activityof the inserted fragment.

Marker genes which have been used for this purpose fall into two maincategories: those which code for proteins which are detected byantibodies and those which code for proteins which are detected by theirenzyme activity. Genes in the first category require the production ofspecific antibodies to the protein and development of a suitabledetection method for measurement of antibody-protein binding. The secondgroup includes genes which code for enzymes such as betagalactosidase,glucuronidase, thymidine kinase, and chloramphenicol acetyltransferase(CAT).

Enzyme marker genes have the advantage of being easy to assay by simplymeasuring enzyme activity in the cellular extract, whileimmunoassay-based systems require the development of specific antibodiesand an immunoassay technique which may be complex and tedious. However,many enzyme assays are subject to background interference by endogenousenzyme activity in the cells being measured. The CAT gene from bacteriais not normally found in mammalian cells and therefore offers thebenefit of a simple, sensitive enzyme-based assay which is free frombackground interference.

Use of the CAT gene as a marker for measuring promoter function intransfected recombinants was first described by Gorman, et. al,"Recombinant Genomes which Express Chloranphenicol Acetyltransferase inMamalian Cells," Mol. Cell Biol. 2:1044 (1982). In this work, CAT enzymeactivity in cell extracts was measured by extracting the [¹⁴C]chloramphenicol mixture into an organic solvent (ethyl acetate) andseparating the mono- and di-acetates by thin-layer chromatography onsilica gel. After autoradiography of the separated chloramphenicolderivatives, the spots were cut from the plates and counted to give aquantitative estimate of CAT activity. Other investigators [Young, et.al., "Detection of Acetyltransferase Activity in Transfected Cells: ARapid and Sensitive HPLC-Based Method," DNA 4(6):469 (1985) and Burzio,et. al., "Assay of Chloramphenicol Acyltransferase by High-PerformanceLiquid Chromatography, Gene Anal. Techn. 5:5 (1988)]have measured CATactivity by HPLC of the organic extract and claim sensitivity equivalentto the original TLC method with the added advantage of a substantialreduction in processing time and the elimination of radioactivematerials. A third method for assaying CAT was reported by Neumann, et.al., "A Novel Rapid Assay for Chloramphenicol Acetyltransferase GeneExpression," Biotechniques 5(5):444 (1987). Using [³ H]acetyl Coenzyme A(which transfers acetyl to chloramphenicol in the enzymic process), thecell incubation was performed in a scintillation vial overlaid withscintillation cocktail. Under these conditions, the only radioactiveproduct that diffuses into the cocktail is acetylated chloramphenicol,allowing kinetic analysis through direct measurement of theradioactivity without requiring a manual separation step. This method isfast and convenient, but requires expensive and hazardous radioactivematerials of high specific activity.

The use of fluorescence as a means of detection in enzyme assays andimmunoassays is recognized to provide many advantages over methods thatemploy spectrophotometry or measurement of radioactivity for detection[Gerson, "Fluorescence Immunoassay," J. Clin. Immunoassay 7(1):73(1984)]. Among these advantages is the elimination of the danger andexpense of handling radioactive materials and the higher potentialsensitivity afforded by measurement of a fluorescent signal as comparedto spectrophotometry. Higher sensitivity provides, in turn, thepotential advantages of greater accuracy and faster assay times.

SUMMARY OF THE INVENTION

In the determination of CAT enzyme activity, the fluorescentchloramphenicol derivative is acylated in the presence of CAT to producefluorescent mono- and diacylchloramphenicol derivatives which are thenseparated by thin layer chromatography (TLC), high performance liquidchromatography (HPLC) or other suitable means and quantitated bymeasurement of their fluorescence and/or absorbance.

Although fluorescent chloramphenicol derivatives have been reported foruse in immunoassays [U.S. Pat. No. 4,420,568 (Wang et al. 1983)], asbactericidal dyes [Soviet Union Patent No. 392,716 (Liverant)] and indrug detection [Nowicki, "Studies on Fluorescamine. Part II--Thin-layerChromatographic Mobilities of Fluorescamine Positive Drugs," AnalyticalLetters 12(A9):1019 (1979)], no fluorescent derivatives ofchloramphenicol have been described which have been used as substratesfor CAT. In order to be useful for this purpose, a fluorescentchloramphenicol derivative must be soluble in aqueous media nearphysiological pH, act as an effective substrate for the enzyme and haveacylation products with high fluorescence efficiency which can be easilyseparated from the starting material.

Fluorescent chloramphenicol derivatives which are the subject of thisinvention exhibit spectral properties which are, for the most part,comparable to fluorescein or 7-dimethylaminocoumarin, both of which havehigh absorbance and display fluorescence with high quantum efficiency.The subject compounds are soluble in aqueous media at pH 7.0-8.0 atconcentrations greater than the K_(m) (the Michlis-Menton constant orthe concentration at half maximum velocity) for chloramphenicol (˜15.0μmolar) reported by Shaw, et. al., "The Enzymatic Acetylation ofChloramphenicol by Extracts of R Factor-Resistant Escherichia coli," J.Biol. Chem. 242(4):687 (1967). Most of the subject compounds are activeas substrates for CAT, some exhibiting velocities greater than 60% ofchloramphenicol. The fluorescent mono- and di-acyl derivatives of thesubject compounds are easily separable from the non-acylated substratesby TLC and HPLC, allowing precise quantitation of their concentration inthe assay mixture by fluorescence spectroscopy.

An example of an application of this invention is the assay of AIDSvirus and human immunodeficiency virus (HIV-1) [Felber, et. al, "AQuantitive Bioassay for HIV-1 Based on Trans-Activation," Science239:184 (1988)]or other viruses.

It is therefore the object of this invention to provide fluorescentderivatives of chloramphenicol for use in the detection and quantitationof enzymes which utilize chloramphenicol or its analogues as substrate.These enzymes include, but are not limited to chloramphenicolacetyltransferase (CAT).

DESCRIPTION OF THE DRAWING

In the drawing:

FIG. 1 is a photograph of a plate identifying CAT in cellular extracts.

DETAILED DESCRIPTION

The subject invention concerns reagents for use in the fluorometricdetermination of chloramphenicol acetyltransferase activity and methodsfor their use. Specifically, the reagents are fluorescent derivatives ofchloramphenicol which are synthesized by reaction ofD(-)threo-2-amino-1-(p-nitrophenyl)-1,3-propanediol (chloramphenicolbase) or other suitable derivatives of chloramphenicol base with asuitably activated fluorescent molecule.

The chloramphenicol base derivative will normally contain a nitrogenattached to the aromatic ring at the paraposition; however other typesof substituents at various locations on the aromatic ring, such asalkyl-, hydroxy-, alkoxy-, aryl-, or halo-will also be suitable for theintended use. The fluorescent moiety will normally be separated from theamine of the chloramphenicol base derivative by a chain of at least twoand not more than twelve atoms other than hydrogen, the preferred numberof atoms depending on the nature of the aryl substituent on thechloramphenicol base derivative. For example, a nitro- or amino- arylsubstituent causes the fluorescence intensity of the fluorophore to besignificantly reduced when the number of atoms in the linking chain isless than about six atoms. Increasing the length of the linking chain tosix to seven atoms results in sufficient separation between the nitro-or amino- substituent and the fluorophore that the fluorescenceintensity is not significantly reduced. The atoms in the linking chainmay include, but are not necessarily limited to carbon, nitrogen, oxygenor sulfur.

When the linking chain contains a carbonyl group adjacent to the amineof the chloramphenicol base derivative, the bond will usually be anamide bond, formed by reaction of the amine with an activated carboxyderivative of the fluorescent molecule, or a urethane bond, formed byreaction of the amine with an activated carbonate derivative of thefluorescent molecule, or a urea bond, formed by reaction of the aminewith an isocyanate derivative of the fluorescent molecule. When thelinking chain contains thiocarbonyl adjacent to the amine of thechloramphenicol base derivative, the bond will usually be a thioureabond, formed by reaction of the amine with an isothiocyanate derivativeof the fluorescent compound. When the linking chain contains a sulfonyladjacent to the amine of the chloramphenicol base derivative, the bondwill usually be a sulfonamide bond, formed by reaction of the amine withan activated sulfonic acid derivative of the fluorescent compound. Whenthe linking chain contains a methylene or methine group adjacent to theamine of the chloramphenicol base derivative, the bond will usually beformed by reductive amination of an aldehyde derivative of thefluorescent molecule or by nucleophilic displacement of a labilesubstituent such as halogen. These activated derivatives can be preparedin a variety of ways, which are known to persons skilled in chemistry,from carboxylic acid, hydroxyl, amine or sulfonic acid groups on thefluorescent molecule.

Unique fluorescent derivatives of chloramphenicol, according to thisinvention, have the general structure of formula (1): ##STR2## wherein:

R₁ -R₅, which may be the same or different, are alkyl-, hydroxy-,alkoxy-, aryl-, halo-, nitro-, amino-, alkylamido-, or arylamido; and

X is a fluorophore derived from fluorescein, rhodamine, coumarin,dimethylaminonaphthalene sulfonic acid (dansyl), pyrene, anthracene,nitrobenzoxadiazole (NBD), acridone or dipyrrometheneboron difluoride.

Particularly useful compounds are those of formula (2): ##STR3##wherein:

R is an alkyl-, hydroxy-, alkoxy,- aryl-, or halo- group; or

R is a nitrogen derivative which includes nitro-, amino-, alkylamido-and arylamido-, where the aryl group is a phenyl or alkyl-substitutedphenyl group.

X is a fluorescent derivative which has been attached to the nitrogenatom through a suitable covalent bond, wherein the covalent bond betweenthe amine of the chloramphenicol base derivative and the fluorescentderivative is amide, urethane, urea, thiourea, sulfonamide or alkyl.

X is a fluorescent derivative which is separated from the amine of thechloramphenicol base derivative by a linking chain of from 1 to 12 atomsother than hydrogen, wherein the atoms in the chain are carbon,nitrogen, oxygen or sulfur.

X may be selected from, but is not necessarily limited to, the groupwhich includes derivatives of fluorescein, rhodamine, coumarin,dimethylaminonaphthalene sulfonic acid (dansyl), pyrene, anthracene,nitrobenzoxadiazole (NBD), acridine and dipyrrometheneboron difluoride.

Preferred R groups include alkylamido- (RCONH--), wherein the alkylchain consists of from one to three carbon atoms.

Preferred linking chains between the fluorophore and the aliphaticnitrogen atom of the chloramphenicol base consist of two to four atomsother than hydrogen if R is alkylamido-, and six to twelve atoms otherthan hydrogen if R is nitro- or amino-.

Preferred X groups include derivatives of dipyrrometheneboron difluorideand derivatives of coumarin.

The choice of fluorophore will vary depending on compatibility with theCAT enzyme, extinction coefficient, quantum yield, desired wavelength ofmeasurement, solubility in both aqueous and non-aqueous environments,and ease of separation of the acylation products from starting material.In order to achieve the highest sensitivity, a primary requirement is topreserve the highest possible substrate activity (compared tochloramphenicol). Other factors which contribute to high sensitivity area high extinction coefficient and high quantum yield. Good solubility ofthe substrate in the enzymatic assay solution is desirable so that theconcentration can be maintained at or above the K_(m) of the enzyme(typically around 10 micromolar), while at the same time, goodsolubility of the acylation products in non-aqueous environmentfacilitates extraction of the acylation products prior to separation andextraction of the separated products from the separation medium (usuallysilica-gel TLC plates, as described above and in Example 48). Examplesof fluorescent chloramphenicol derivatives which are the subject of thisinvention are given in Tables I to VIII.

                  TABLE I                                                         ______________________________________                                        FLUORESCENT CHLORAMPHENICOL                                                   ACETYLTRANSFERASE SUBSTRATES                                                  Chloramphenicol-dipyrrometheneboron difluoride Derivatives                    Compound                                                                              R           X                                                         ______________________________________                                        1       Acetamido-  1-(4,4'-Difluoro-3,5,7-trimethyl-4-                                           bora-3a,4a-diaza-s-indaceneacetyl)-                                           (1F)                                                      2       Amino-      1F                                                        3       Nitro-      1F                                                        4       Nitro-      4-((1-(4,4'-Difluoro-3,5,7-trimethyl-                                         4-bora-3a,4a-diaza-s-indaceneacetyl)                                          amino)butyryl)-(2F)                                       5       Amino-      2F                                                        6       Acetamido-  2F                                                        7       3-Carboxypro-                                                                             2F                                                                pionylamino-                                                          8       Acetamido-  2-(4,4'-Difluoro-1,3,5,7-tetramethyl-                                         4-bora-3a,4a-diaza-s-                                                         indacenepropionyl)-(3F)                                   9       Amino-      3F                                                        10      Nitro-      3F                                                        11      Nitro-      4-((2-(4,4'-Difluoro-1,3,5,7-tetra-                                           methyl-4-bora-3a,4a-diaza-s-                                                  indacene propionyl)amino)butyryl)-                                            (4F)                                                      12      Acetamido-  3-(4,4'-Difluoro-5,7-dimethyl-4-                                              bora-3a,4a-diaza-s-indacene-                                                  propionyl)-(5F)                                           13      Amino-      5F                                                        14      Nitro-      5F                                                        15      Acetamido-  4-((3-(4,4'-Difluoro-5,7-dimethyl-4-                                          bora-3a,4a-diaza-s-indacene-                                                  propionyl)amino)butyryl)-(6F)                             16      3-Carboxypro-                                                                             6F                                                                pionylamino-                                                          17      Nitro-      6F                                                        ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        FLUORESCENT CHLORAMPHENICOL                                                   ACETYLTRANSFERASE SUBSTRATES                                                  Chloramphenicol-coumarin Derivatives                                          Compound                                                                              R           X                                                         ______________________________________                                        18      Acetamido-  4-(7-dimethylaminocoumarinacetyl)-                                            (7F)                                                      19      Amino-      7F                                                        20      Nitro-      7F                                                        21      Nitro-      4-((4-(7-dimethyaminocoumarin-                                                acetyl)amino)butyryl)-(8F)                                22      Amino-      8                                                         23      Acetamido-  8F                                                        24      3-Carboxypro-                                                                             8F                                                                pionylamino-                                                          25      Acetamido-  4-((3-(4-methyl-7-                                                            hydroxycoumarinacetyl)                                                        amino)butyryl)-(9F)                                       26      Amino-      9F                                                        27      Nitro-      9F                                                        ______________________________________                                          T1 TABLE III-FLUORESCENT CHLORAMPHENICOL? -ACETYLTRANSFERASE SUBSTRATES?     -Chloramphenicol-NBD Derivatives? -Compound? R? X? -28 Acetamido-     6-((4-(7-nitrobenz-2-oxa-1,3-diazolyl) -  amino)hexanoyl)-(10F) -29 Amino-     10F? -

                  TABLE IV                                                        ______________________________________                                        FLUORESCENT CHLORAMPHENICOL                                                   ACETYLTRANSFERASE SUBSTRATES                                                  Chloramphenicol-pyrene Derivatives                                            Compound    R         X                                                       ______________________________________                                        30          Acetamido-                                                                              1-pyrenebutyryl-(11F)                                   ______________________________________                                    

                  TABLE V                                                         ______________________________________                                        FLUORESCENT CHLORAMPHENICOL                                                   ACETYLTRANSFERASE SUBSTRATES                                                  Chloramphenicol-anthracene Derivatives                                        Compound   R          X                                                       ______________________________________                                        31         Acetamido- 9-anthracenepropionyl-(12F)                             ______________________________________                                    

                  TABLE VI                                                        ______________________________________                                        FLUORESCENT CHLORAMPHENICOL                                                   ACETYLTRANSFERASE SUBSTRATES                                                  Chloramphenicol-acridone Derivatives                                          Compound    R           X                                                     ______________________________________                                        32          Acetamido-  10-acridoneacetyl-(13F)                               33          Amino-      13F                                                   ______________________________________                                    

                  TABLE VII                                                       ______________________________________                                        FLUORESCENT CHLORAMPHENICOL                                                   ACETYLTRANSFERASE SUBSTRATES                                                  Chloramphenicol-dansyl Derivatives                                            Compound R          X                                                         ______________________________________                                        34       Acetamido- 4-((1-(5-dimethylaminonaphthalene                                             sulfonyl)amino)butyryl)-(14F)                             ______________________________________                                    

                  TABLE VIII                                                      ______________________________________                                        FLUORESCENT CHLORAMPHENICOL                                                   ACETYLTRANSFERASE SUBSTRATES                                                  Chloramphenicol-xanthene Derivatives                                          Compound                                                                              R         X                                                           ______________________________________                                        35      Acetamido-                                                                              4-(5-(tetramethylrhodaminethioureidyl)                                        butyryl)-(15F)                                              36      Acetamido-                                                                              4-(6-(fluoresceinthioureidyl)butyryl)-                                        (16F)                                                       ______________________________________                                    

Coumarins and dipyrrometheneboron difluoride fluorophores covalentlybonded to chloramphenicol base or suitable derivatives ofchloramphenicol base are particularly suitable as CAT substrates becausethey show superior activity as substrates, have generally highextinction coefficients and quantum yields and are readily soluble inboth aqueous and non-aqueous solvents. By way of example, Compound 6 hasfluorescence comparable to the corresponding fluorescein derivative 36,but is much more soluble in ethyl acetate, which is a preferredextraction solvent.

As previously discussed, the subject compounds are useful inquantitating chloramphenicol acetyltransferase activity in cellextracts. In order to perform these assays, there must be a method foraccurately and precisely determining the quantity of substrateacetylated during a given time period. Measurement of the acetylationproducts of the subject compounds by their intrinsic fluorescence offersa practical alternative to the currently used radiographic andspectrophotometric procedures described above. Since fluorometry is amuch more sensitive technique than absorptiometry, use of the subjectcompounds will result in an assay with superior sensitivity. Inaddition, the assay will take less time to complete than the tediousradiometric methods. Fluorescence measurements can be carried outdirectly on TLC plates by means of an incident-light fluorometer, on TLCextracts or on the effluent of a HPLC column. Although the measurementof fluorescence offers superior sensitivity when compared to measurementof absorption, it is recognized that the subject compounds for the mostpart have high extinction coefficients so that, in many applications,quantitation by measurement of their absorption will provide an assaywith adequate sensitivity. Absorption measurements can be carried out onTLC extracts or on the effluent of a HPLC column.

The following illustrations describe the practice of the invention andare by way of example and not by way of limitation.

EXAMPLE 1 D(-)threo-2-amino-1-(p-aminophenyl)-1,3-propanediol (CompoundA)

To a suspension of 1.0 g chloramphenicol base in 30 ml methanol wasadded 0.1 g 10% palladium on charcoal. The mixture was pressurized to 50psi with hydrogen and shaken for one hour in a Parr hydrogenationapparatus at room temperature. The reaction mixture was filtered througha diatomateous earth pad and the filtrate was concentrated on a rotaryevaporator to give a pale yellow solid. Recrystallization from methanolyielded 450 mg of off-white crystals. A second crop of 300 mg ofcrystals was obtained from the mother liquor after cooling in arefrigerator overnight. Total yield was 0.75 g Compound A, which waschromatographically homogeneous.

EXAMPLE 2D(-)threo-2-(N-t-BOC-(4-aminobutyrylamino))-1-(p-nitrophenyl)-1,3-propanediol(Compound B)

To a mixture of 0.5 g chloramphenicol base, 0.48 gN-t-BOC-3-aminobutyric acid, 0.32 g hydroxybenzotriazole and 420 μLdiisopropylethylamine in 20 mL THF was added 0.49 g N,N-dicyclohexylcarbodiimide. The reaction mixture was stirred at room temperature for18 hours. The solution was filtered from precipitated dicyclohexylureaand the filtrate was evaporated on a rotary evaporator to give a yellowoil. The crude product was purified on a 2×35 cm silica gel column,eluting first with 2% methanol in chloroform, then with 5% methanol inchloroform. Evaporation of the major fraction yielded 0.75 g Compound B,isolated as a white gum-like solid which was homogeneous by TLC.

EXAMPLE 3D(-)threo-2-(4-aminobutyrylamino)-1-(p-nitrophenyl)-1,3-propane-diol(Compound C)

Trifluoroacetic acid (10 mL) was added to 3.5 g Compound B, and themixture was stirred at room temperature for 20 minutes. The solution wasthen poured into 100 mL ether with vigorous stirring and the precipitatewas collected by filtration and washed several times with ether. Thehygroscopic product was dried overnight in a vacuum desiccator to yield3.1 g Compound C, which was chromatographically homogeneous.

EXAMPLE 4D(-)threo-2-(N-t-BOC-(4-aminobutyrylamino))-1-(p-aminophenyl)-1,3-propanediol(Compound D)

To 3.0 g Compound B in 100 mL methanol was added 300 mg 10% palladium oncharcoal. Hydrogenation was carried out as described in Example 1 for 30minutes to yield 2.6 g Compound D.

EXAMPLE 5D(-)threo-2-(N-t-BOC-(4-aminobutyrylamino))-1-(p-acetamidophenyl)-1,3-propanediol(Compound E)

To a solution of 1.5 g Compound D in 40 mL dry THF was added 400 μLacetic anhydride and the mixture was stirred at room temperature for 3hours. The resulting reaction mixture was concentrated on a rotaryevaporator and the residual oil was purified by silica gel columnchromatography (2×25 cm column), eluting with 10% methanol inchloroform. After removal of the solvent from the major fraction, ayield of 1.4 g of the desired product, Compound E, was obtained.

EXAMPLE 6D(-)threo-2-(4-aminobutyrylamino)-1-(p-acetamidophenyl)-1,3-propanediol(Compound F)

To 1.4 g Compound E was added 5 mL trifluoroacetic acid. After stirringat room temperature for 30 minutes, the reaction mixture was worked upas described in Example 3 to yield 0.9 g Compound F.

EXAMPLE 7D(-)threo-2-(N-t-BOC-(4-aminobutyrylamino))-1-(p-(3-carboxypropionylaminophenyl))-1,3-propanediol(Compound G)

To a solution of 0.5 g Compound D was added 0.14 g succinic anhydride.The solution was stirred at room temperature for 8 hours andconcentrated on a rotary evaporator to yield a crude oil. This crudeproduct was subjected to silica gel column chromatography (2×15 cmcolumn), eluting with 20% methanol in chloroform. The yield of theproduct, Compound G, was 0.32 g.

EXAMPLE 8D(-)threo-2-(4-aminobutyrylamino)-1-(p-(3-carboxypropionylaminophenyl))-1,3-propanediol(Compound H)

Trifluoroacetic acid (10 mL) was added to 0.32 g Compound G. The mixturewas stirred 30 minutes at room temperature and worked up as described inExample 3 to yield 190 mg Compound H.

EXAMPLE 9D(-)threo-2-(4-aminobutyrylamino)-1-(p-aminophenyl)-1,3-propanediol(Compound I)

To a sample of 0.69 q Compound D was added 3 mL trifluoroacetic acid.After stirring at room temperature for 30 minutes, the reaction mixturewas worked up as described in example 3 to yield to yield 0.45 gCompound I.

EXAMPLE 10 N-2-(D(-)threo-1-(paminophenyl)-1,3-dihydroxypropyl)-4,4'-difluoro-3,5,7-trimethyl-4-bora-3a,4a-diaza-s-indacene-1-acetamide(Compound 2)

To a suspension of 10 mg Compound A in 5 mL dry DMF was added 25 μLtriethylamine. After stirring at room temperature for a few minutes, 21mg of the succinimidyl ester of Compound 1F was added. The mixture wasstirred overnight at room temperature. The solution was concentrated ona rotary evaporator and the residue was purified by silica-gelchromatography (1×20 cm column), eluting with 5% methanol in chloroform.The major fractions were combined and evaporated to yield 14 mg Compound2.

EXAMPLE 11N-2-(D(-)threo-1-(p-acetamidophenyl)-1,3-dihydroxypropyl)-4,4'-difluoro-3,5,7-trimethyl-4-bora-3a,4a-diaza-s-indacene-1-acetamide(Compound 1)

Compound 2 (8 mg) was dissolved in 2 mL acetone and 5 drops aceticanhydride was added. The solution was stirred at room temperature for 1hour, then concentrated on a rotary evaporator. The crude product waspurified by silica-gel chromatography (1×15 cm column) as in Example 10,to yield 8 mg Compound 1.

EXAMPLE 12N-2-(D(-)threo-1-(p-aminophenyl)-1,3-dihydroxypropyl)-4,4'-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-2-propionamide(Compound 9)

To a suspension of 10 mg Compound A in 5 mL dry DMF was added 25 μLtriethylamine. After stirring at room temperature for a few minutes, thesuccinimidyl ester of Compound 3F was added and the solution was stirredat room temperature overnight. The reaction mixture was worked up asdescribed in Example 10 giving 20 mg Compound 9.

EXAMPLE 13N-2-(D(-)threo-1-(p-acetamidophenyl)-1,3-dihydroxypropyl)-4,4'-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-2-propionamide(Compound 8)

10 mg Compound 9 in 2 mL acetone was added 5 drops acetic anhydride andthe mixture was stirred for 1 hour at room temperature. The solvent wasremoved on a rotary evaporator and the residual solid was dissolved in aminimum amount of DMF and chromatographed on a 1×20 cm silica-gelcolumn, eluting with 8% methanol in chloroform. The major fraction wascombined and the solvent evaporated to yield 10 mg Compound 8.

EXAMPLE 14N-2-(D(-)threo-1-(p-aminophenyl)-1,3-dihydroxypropyl)-4,4'-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionamide(Compound 13)

Triethylamine (25 μL) was added to a suspension of 10 mg Compound A in 2mL dry THF and the mixture was stirred at room temperature for a fewminutes. Then 21 mg of the succinimidyl ester of Compound 5F was addedand the solution was stirred at room temperature overnight. Work-up ofthe crude reaction mixture was performed as described in Example 10,using a 1×10 cm silica-gel column, to yield 20 mg Compound 13.

EXAMPLE 15N-2-(D(-)threo-1-(p-acetamidophenyl)-1,3-dihydroxypropyl)-4,4'-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionamide(Compound 12)

Two drops acetic anhydride was added to a solution of 15 mg Compound 13in 0.5 mL acetone and the mixture was stirred at room temperature for 1hour. After addition of 1 mL of methanol to quench the reaction, thesolvent was removed on a rotary evaporator and the residue was purifiedon a 2×10 cm silica-gel column, eluting with 5% methanol in chloroformto yield 12 mg of Compound 12.

EXAMPLE 164-(N-2-(D(-)threo-1-(p-acetamidophenyl)-1,3-dihydroxypropyl)-(3-(4,4'-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacenepropionyl)amino)butyramide(Compound 15)

To a solution of 17 mg Compound F in 0.2 mL dry DMF was added 10 μL oftriethylamine. Then 15 mg of the succinimidyl ester of Compound 5F in 1mL dry DMF was added and the mixture was stirred at room temperatureovernight. Work-up of the crude reaction mixture, as described inExample 10, using a 1.5 cm×5 cm silica-gel column gave 13 mg of Compound15.

EXAMPLE 174-(N2-(D(-)threo-1-(p-aminophenyl)-1,3-dihydroxypropyl)-(1-(4,4'-difluoro-3,5,7-trimethyl-4-bora-3a,4a-diaza-s-indaceneacetyl)amino)butyramide(Compound 5)

To a solution of 16 mg of Compound I in 0.5 mL dry DMF was added 2.0 mLDry THF. Then 10 μL of triethylamine was added, followed by 15 mg of thesuccinimidyl ester of Compound 1F. The reaction mixture was stirred atroom temperature overnight. Work-up of the crude reaction mixture wasaccomplished as described in Example 10, using a 1.5×20 cm silica-gelcolumn, eluting with 8% methanol in chloroform, gave 11 mg of Compound5.

EXAMPLE 184-(N-2-(D(-)threo-1-(p-(3-carboxypropionylaminophenyl))-1,3-dihydroxypropyl)-(1-(4,4'-difluoro-3,5,7-trimethyl-4-bora-3a,4a-diaza-s-indaceneacetyl)amino)butyramide(Compound 7)

To a suspension of 6.0 mg of Compound 5 in 5 mL of chloroform was added1.1 mg of succinic anhydride. The mixture was stirred overnight. Aprecipitate formed, which was filtered and washed thoroughly withchloroform TLC showed the presence of a single product. The precipitatewas dried under vacuum at room temperature to yield 5 mg of Compound 7.

EXAMPLE 194-(N-2-(D(-)threo-1-(p-acetamidophenyl)-1,3-dihydroxypropyl)-(1-(4,4'-difluoro-3,5,7-trimethyl-4-bora-3a,4a-diaza-s-indaceneacetyl)amino)butyramide(Compound 6)

To a solution of 17 mg of Compound F in 0.5 mL of dry DMF was added 3 mLof dry THF. Then, 10 μL of triethylamine was added, followed by 15 mg ofCompound IF. The mixture was stirred overnight at room temperature. Thecrude reaction mixture was chromatographed on a 1×20 cm silica-gelcolumn as described in Example 10, using 8% methanol in chloroform toyield 14 mg of Compound 6.

EXAMPLE 204-(N-2-(D(-)threo-1-(p-(3-carboxypropionylaminophenyl))-1,3-dihydroxypropyl)-(3-(4,4'-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacenepropionyl)amino)butyramide(Compound 16)

To a solution of 13 mg Compound H in 1 mL dry DMF was added 10 μL oftriethylamine and 10 mg of the succinimidyl ester of Compound 5F. Thereaction mixture was stirred at room temperature overnight and theproduct isolated by silica-gel chromatography as described in Example10, using 10% methanol in methylene chloride. The yield of Compound 16was 14 mg.

EXAMPLE 21N-2-(D(-)threo-1-(p-nitrophenyl)-1,3-dihydroxypropyl)-3-(4,4'-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacenepropionamide(Compound 17)

To a solution of 10 mg of chloramphenicol base in 1 mL of dry THF wasadded 10 μL of triethylamine, followed by 18 mg of the succinimidylester of Compound 5F. The reaction mixture was stirred at roomtemperature overnight. The reaction mixture was concentrated and theresidue was purified by column chromatography (1.5×10 cm silica-gel),first eluting with 2% methanol in methylene chloride until the minorimpurities were removed, then with 7% methanol in methylene chloride.The yield of Compound 17 was 19 mg.

EXAMPLE 22N-2-(D(-)threo-1-(p-nitrophenyl)-1,3-dihydroxypropyl)-1-(4,4'-difluoro-3,5,7-trimethyl-4-bora-3a,4a-diaza-s-indaceneacetamide(Compound 3)

To a solution of 14 mg of chloramphenicol base and 10 μL oftriethylamine in 1 mL of dry THF was added 25 mg of the succinimidylester of Compound 1F and the mixture was stirred at room temperatureovernight. The solvent was removed on a rotary evaporator and theresidual oil was purified by preparative TLC on 20×20 cm x 250 micronsilica-gel plates. The plates were eluted 3 times with 3% methanol inchloroform, and the major band was extracted with chloroform. Afterevaporation of the solvent, TLC showed a small amount of impurity. Theoil was dissolved in 10 mL of chloroform and extracted with 5% sodiumbicarbonate solution. The chloroform layer was dried over sodium sulfateand evaporated under vacuum to yield 22 mg of Compound 3.

EXAMPLE 234-(N-2-(D(-)threo-1-(p-nitrophenyl)-1,3-dihydroxypropyl)-(1-(4,4'-difluoro-3,5,7-trimethyl-4-bora-3a,4a-diaza-s-indaceneacetyl)amino)butyramide(Compound 4)

To a solution of 11 mg of Compound C in 0.5 mL of dry DMF was added 25μL of triethylamine followed by 10 mg of the succinimidyl ester ofCompound IF. The reaction mixture was stirred at room temperatureovernight and the solvent was removed under vacuum to yield a red-orangesolid. The crude product was purified as described in Example 10 toyield 11 mg of Compound 4.

EXAMPLE 24N-2-(D(-)threo-1-(p-nitrophenyl)-1,3-dihydroxypropyl)-2-(4,4'-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacenepropionamide)(Compound 10)

To a suspension of 10 mg of chloramphenicol base in 3 mL of dry THF wasadded 10 μL of triethylamine. The suspension was stirred several minuteswhereupon all the solid material was completely dissolved. Then 15 mg ofthe succinimidyl ester of Compound 3F was added and the resultingmixture was stirred at room temperature for two days. The crude reactionmixture was concentrated under vacuum and the oily residue was purifiedby preparative TLC on silica-gel plates as described in Example 22 toyield 18 mg of Compound 10.

EXAMPLE 254-(N-2-(D(-)threo-1-(p-nitrophenyl)-1,3-dihydroxypropyl)-(2-(4,4'-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacenepropionyl)amino)butyramide(Compound 11)

To a solution of 10 mg of Compound C in 0.5 mL of dry THF was added 25μL of triethylamine followed by 10 mg of the succinimidyl ester ofCompound 3F and the mixture was stirred at room temperature for fourdays. The solvent was evaporated and the crude product waschromatographed on a 1×20 cm silica-gel column, eluting with chloroformto yield 12 mg of Compound 11.

EXAMPLE 264-(N-2-(D(-)threo-1-(p-nitrophenyl)-1,3-dihydroxypropyl)-(3-(4,4'-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacenepropionyl)amino)butyramide(Compound 14)

To a suspension of 20 mg of Compound C in 3 mL of dry THF was added 10μL of triethylamine, followed by the addition of 19 mg of thesuccinimidyl ester of compound 5F. The reaction mixture was stirredovernight at room temperature and the solvent was removed under vacuum.The crude product was purified by column chromatography (1.5×10 cm) onsilica-gel as described in example 21 to yield 18 mg Compound 14.

EXAMPLE 27N-2-(D(-)threo-1-(p-aminophenyl)-1,3-dihydroxypropyl)-7-dimethylaminocoumarin-4-acetamide(Compound 19)

To a suspension of 25 mg of the succinimidyl ester of Compound 7F in 5mL of dry THF was added one drop of triethylamine and 14 mg of CompoundA. The reaction mixture was stirred at room temperature overnight,filtered and the solvent removed under vacuum. The resulting crudeproduct was purified by silica-gel column chromatography (1×20 cm),eluting with 5% methanol in chloroform. A yield of 23 mg of Compound 19was obtained.

EXAMPLE 28N-2-(D(-)threo-1-(p-acetamidophenyl)-1,3-dihydroxypropyl)-7-dimethylaminocoumarin-4-acetamide(Compound 18)

To a suspension of Compound 19 in 2 mL of acetone was added five dropsof acetic anhydride and the mixture was stirred at room temperature for2 hours. The reaction mixture was still heterogeneous, so 0.5 mL of dryDMF and an additional 3 drops of acetic anhydride was added. After atotal of 3 hours reaction time, the crude reaction mixture was reducedunder vacuum and the residual DMF solution was poured into 30 mL ofether. The pale yellow solid was collected by filtration and the crudeproduct was purified by silica-gel column chromatography (1×15 cm). Thecrude product was dissolved in a minimum amount of DMF and then loadedon the column and eluted with 5% methanol in chloroform. In this manner,9 mg of Compound 18 was obtained.

EXAMPLE 29N-2-(D(-)threo-1-(p-nitrophenyl)-1,3-dihydroxypropyl)-7-dimethylaminocoumarin-4-acetamide(Compound 20)

To a solution of 20 mg of the succinimidyl ester of compound 7F in 2 mLof dry THF was added 20 μL of triethylamine and 13 mg of chloramphenicolbase. The reaction mixture was stirred at room temperature for 6 hoursand the precipitate was filtered. The product, Compound 20 washomogeneous by TLC and therefore needed no further purification.

EXAMPLE 304-(N-2-(D(-)threo-1-(p-nitrophenyl)-1,3-dihydroxypropyl)-(4-(7-dimethylaminocoumarinacetyl)amino)butyramide(Compound 21)

To a solution of 24 mg of Compound C in 1 mL of dry THF was added 3drops of triethylamine, followed by the addition of 20 mg of thesuccinimidyl ester of Compound 7A. The reaction mixture was stirred atroom temperature overnight. The resulting precipitate was filtered offand purified by column chromatography on silica-gel (2×15 cm), elutingwith 5% ethanol in chloroform. A yield of 7 mg of Compound 21 wasobtained.

EXAMPLE 314-(N-2-(D(-)threo-1-(p-aminophenyl)-1,3-dihydroxypropyl)-(4-(7-dimethylaminocoumarinacetyl)amino)butyramide(Compound 22)

A sample of 29 mg of Compound I was dissolved in 0.5 mL of dry DMF. Tothis solution was added 15 μL of triethylamine and 25 mg of thesuccinimidyl ester of Compound 7F and the reaction mixture was stirredovernight. The solvent was removed under vacuum and the residue waspurified by column chromatography on silica-gel (2×15 cm), eluting with10% methanol in chloroform. Three fractions were obtained. The firstfraction contained mainly a single product, the second and thirdfractions contained the product of the first fraction and a secondproduct which had a lower Rf value (silica-gel TLC). The second andthird fractions were combined and chromatographed again under the sameconditions. A yield of 0.2 mg of Compound 22 was obtained.

EXAMPLE 324-(N-2-(D(-)threo-1-(p-acetamidophenyl)-1,3-dihydroxypropyl]-(4-(7-dimethylaminocoumarinacetyl)amino)butyramide(Compound 23)

To a solution of 65 mg of Compound F in 12 mL of 10% DMF in THF wasadded 30 μL of triethylamine and 50 mg of the succinimidyl ester ofCompound 7F. The reaction mixture was stirred at room temperatureovernight. The resulting precipitate was filtered and the solid waswashed several times with dry THF. After drying, 70 mg of Compound 23was obtained.

EXAMPLE 334-(N-2-(D(-)threo-1-(p-(3-carboxypropionylaminophenyl))-1,3-dihydroxypropyl)-(4-(7-dimethylaminocoumarinacetyl)amino)butyramide(Compound 24)

A sample of 42 mg of Compound H was dissolved in 3 mL of dry 1:3 DMF inTHF. Into the above solution was added 20 μL of triethylamine followedby 30 mg of the succinimidyl ester of 7F.

The reaction mixture was stirred at room temperature overnight,concentrated under vacuum and the residue purified by silica-gel columnchromatography (2×10 cm column). elution was carried out with 0.2%acetic acid, 20% methanol in chloroform. A yield of 17 mg of Compound 24was obtained.

EXAMPLE 344-(N-2-(D(-)threo-1-(p-acetamidophenyl)-1,3-dihydroxypropyl)-(3-(4-methyl-7-hydroxycoumarinacetyl)amino)butyramide(Compound 25)

To a solution of 13 mg of Compound F in 3 mL of 1:3 dry DMF in THF wasadded 10 μL of triethylamine and 10 mg of the succinimidyl ester of4-methylcoumarin-3-acetic acid. The reaction mixture was stirred at roomtemperature overnight, concentrated under vacuum and the residue waspurified by silica-gel column chromatography (2×10 cm column), elutingwith 5% methanol in chloroform. The yield of Compound 25 was 9 mg.

EXAMPLE 354-(N-2-(D(-)threo-1-(p-nitrophenyl)-1,3-dihydroxypropyl)-(3-(4-methyl-7-hydroxycoumarinacetyl)amino)butyramide(Compound 26)

To a solution of 13 mg of Compound C in 2 mL of dry DMF was added 10 μLof triethylamine, followed by the addition of 10 mg of the succinimidylester of 4-methylcoumarin-3-acetic acid. The reaction mixture wasstirred at room temperature overnight, concentrated under vacuum and theresidue was purified as described in Example 34. The yield of Compound25 was 11 mg.

EXAMPLE 364-(N-2-(D(-)threo-1-(p-aminophenyl)-1,3-dihydroxypropyl)-(3-(4-methyl-7-hydroxycoumarinacetyl)amino)butyramide(Compound 27)

To a solution of 5 mg of Compound 26 in 2 mL of methanol in a test tubewas added a magnetic stir bar and a few mg palladium on charcoal.Hydrogen gas was bubbled through the solution while stirring vigorouslyfor 10 minutes. The reaction mixture was filtered through a diatomaceousearth pad, the solvent was evaporated and the residual solid waspurified by column chromatography (2×10 cm silica-gel column), elutingwith 10% methanol in chloroform. The yield of Compound 27 was 2 mg.

EXAMPLE 376-(N-2-(D(-)threo-1-(p-aminophenyl)-1,3-dihydroxypropyl)-(4-(7-nitrobenz-2-oxa-1,3-diazolyl)amino)hexanamide(Compound 28)

To a solution of 7 mg of Compound A in 2 mL of dry THF was added 10 μLof triethylamine, followed by the addition of 16 mg of The succinimidylester of Compound 10F. The reaction mixture was stirred overnight atroom temperature. The solvent was removed under vacuum and the residuewas purified by silica-gel column chromatography (1×10 cm column),eluting with 5% methanol in methylene chloride, to yield 18 mg ofCompound 28.

EXAMPLE 386-(N-2-(D(-)threo-1-(p-aminophenyl)-1,3-dihydroxypropyl)-(4-(7-nitrobenz-2-oxa-1,3-diazolyl)amino)hexanamide(Compound 29)

To a solution of 12 mg of Compound 28 in 1 mL of dry THF was added onedrop of acetic anhydride and the mixture was stirred at room temperaturefor 1 hour. The 5 mL of methanol was added and the mixture was stirredfor a few minutes. The solvent was removed under vacuum and the residuewas purified by silica-gel column chromatography (1.5×15 cm column),eluting with 5% methanol in dichloromethane, to yield 10 mg of Compound29.

EXAMPLE 39N-2-(D(-)threo-1-(p-aminophenyl)-1,3-dihydroxypropyl)-1-pyrenebutyramide(Compound J)

To a solution of 10 mg of Compound A in 2 mL of dry THF was added 15 μLof triethylamine, followed by the addition of 21 mg of succinimidylpyrene-1-butyrate. The reaction mixture was stirred at room temperatureovernight, reduced to dryness under vacuum and the residue subjected topurification by silica-gel column chromatography (1.5×15 cm column),eluting with 3% methanol in methylene chloride. The yield of Compound Jwas 22 mg.

EXAMPLE 40N-2-(D(-)threo-1-(p-acetamidophenyl)-1,3-dihydroxypropyl)-1-pyrenebutyramid(Compound 30)

To a solution of 20 mg of Compound J in 1 mL of dry THF was added onedrop of acetic anhydride and the mixture was stirred at room temperaturefor 1 hour. Then 0.5 mL of methanol was added and stirring was continuedfor several minutes. The solvent was removed under vacuum and theresidue was subjected to column chromatography on silica-gel (1.5×15 cmcolumn), eluting with 5% methanol in chloroform to yield 15 mg ofCompound 30.

EXAMPLE 41N-2-(D(-)threo-1-(p-aminophenyl)-1,3-dihydroxypropyl)-9-anthracenepropionamide(Compound K)

To a solution of 10 mg of Compound A in 2 mL of dry DMF was added 15 μLof triethylamine, followed by the addition of 20 mg of succinimidylanthracene-9-propionate, and the mixture was stirred at room temperatureovernight. The solvent was removed under vacuum and the residue purifiedon a 1.5×15 cm silica-gel column, eluting with 3% methanol in chloroformto give 19 mg of Compound K, contaminated with an impurity, probablyanthracene-9-propionic acid.

EXAMPLE 42N-2-(D(-)threo-1-(p-acetamidophenyl)-1,3-dihydroxypropyl)-9-anthracenepropionamide(Compound 31)

To a solution of 15 mg of Compound K in 1 mL of dry THF was added onedrop of acetic anhydride and the reaction mixture was stirred at roomtemperature for 1 hour. Then 0.5 mL of methanol was added and stirringwas continued for several minutes. The solvent was removed under vacuumand the residue was purified on a 1.5×15 cm silica-gel column, elutingwith 5% methanol in methylene chloride, to give 7 mg of Compound 31.

EXAMPLE 43N-2-(D(-)threo-1-(p-aminophenyl)-1,3-dihydroxypropyl)-10-acridoneacetamide(Compound 33)

To a solution of 10 mg of Compound A in 2 mL of dry DMF was added 15 μLof triethylamine, followed by the addition of 19 mg of succinimidylacridone-10-acetate and the reaction mixture was stirred at roomtemperature overnight. The resulting white precipitate was collected byfiltration and the solid was washed several times with ether to yield,after drying, 14 mg of Compound 33.

EXAMPLE 44N-2-(D(-)threo-1-(p-acetamidophenyl)-1,3-dihydroxypropyl)-10-acridoneacetamide(Compound 32)

To a solution of 12 mg of Compound 33 in 0.5 mL of dry DMF was added onedrop of acetic anhydride and the mixture was stirred at room temperaturefor one hour. Then 0.5 mL of methanol was added and the mixture wasstirred a few more minutes. The solvent was removed under vacuum and theresidue was purified on a 1.5×15 cm silica-gel column, eluting with 10%methanol in methylene chloride to yield 10 mg of Compound 32.

EXAMPLE 454-(N-2-(D(-)threo-1-(p-acetamidophenyl)-1,3-dihydroxypropyl)-(1-(5-dimethylaminonaphthalenesulfonyl)amino)butyramide(Compound 34)

To a solution of 10 mg of Compound F in 0.5 mL of dry DMF was added 10μL of triethylamine, followed by 7 mg of5-dimethylaminonaphthalenesulfonyl chloride. The reaction mixture wasstirred at room temperature overnight and the solvent was removed undervacuum. The crude product was purified using a 1.5×15 cm silica-gelcolumn, eluting with 10% methanol in methylene chloride. A yield of 8 mgof Compound 34.

EXAMPLE 464-(N-2-(D(-)threo-1-(p-acetamidophenyl)-1,3-dihydroxypropyl)-(5-(tetramethylrhodaminethioureidyl)amino)butyramide(Compound 35)

To a solution of 5 mg of Compound F in 0.5 mL of dry DMF was added 10 μLof triethylamine, followed by the addition of 5 mg oftetramethylrhodamine-5-isothiocyanate and the mixture was stirred atroom temperature overnight. The solvent was removed under vacuum and thecrude product was chromatographed on a 1.5×5 cm silica-gel column,eluting with 20% methanol in methylene chloride, to give 3 mg ofCompound 35.

EXAMPLE 474-(N-2-(D(-)threo-1-(p-acetamidophenyl)-1,3-dihydroxypropyl)-(6-(fluoresceinthioureidyl)amino)butyramide(Compound 36)

To a solution of 10 mg of Compound F in 0.5 mL of dry DMF was added 10μL of triethylamine, followed by 10 mg of fluorescein-6-isothiocyanate.The reaction mixture was stirred at room temperature overnight and thenconcentrated under vacuum. The residue was subjected to silica-gelchromatography (1.5×10 cm column), eluting with 15% methanol indichloromethane, to give 11 mg of Compound 36.

EXAMPLE 48 Procedure for Comparing Activity of Fluorescent Substrates

To a solution of 0.5 mg acetyl CoA in 112 μL of 1M TRIS pH 7.4 was added7 μL of a solution of 440 uM fluorescent CAT substrate in ethanol. Then0.8 U of purified CAT in 1 μL of 1M TRIS (pH 7.4) was added and thesolution was incubated at 37° C. for 1 hour. The reaction mixture wasextracted with 0.7 mL of ethyl acetate, and the solvent was removedunder vacuum. The residue was dissolved in 14 μL of ethyl acetate,spotted on the pre-absorbent area of Whatman LK6 silica-gel TLC platesand developed with 10% methanol in chloroform. A control, run as abovebut with no enzyme present, was spotted adjacent to each reactionmixture.

Qualitative estimates of substrate activity of each fluorescentderivative were made by examination of the plates for conversion toacetylated products. Compounds 6, 21, 23 and 28 appeared to have thehighest reaction velocities of the tested fluorescent substrates.

EXAMPLE 49

Determination of K_(m) for Compound 6

The K_(m) was determined for Compound 6 using a direct linear plot[Eisenthal, et. al., "The Direct Linear Plot. A New Graphical Proceedurefor Estimating Enzyme Kinetic Parameters," Biochem. J. 139:715 (1974)].Four different concentrations of substrate, 1, 8, 12 and 40 μM, wereincubated with 0.8U of purified enzyme and 0.4 mg Acetyl CoA in 0.5MTRIS, pH 7.4 for 2.5 min. The initial concentrations were determinedspectroscopically using the extinction coefficient of Compound 6(76,000). The conversion from substrate to acetylation products wasdetermined by extraction from the TLC separation and measuringfluorescence at 532 nm with excitation at 504 nm. The reaction velocityin picomoles/min was calculated from the concentration. A plot ofreaction velocity vs initial concentration was made for each of the fourdifferent concentrations, assuming the velocity at 2.5 minutes did notdiffer significantly from initial velocity. The K_(m), 2.0 μM, was readfrom the X-axis at the point where the four rate curves intersect.

EXAMPLE 50 Relative Velocity of Compound 6 vs Choramphenicol

The velocity during the first ten minutes at a substrate concentrationof 33.2 μM was determined using Compound 6 and ¹⁴ C chloramphenicol. Thereactions were run in 100 μL final volume TRIS pH 7.4 with 0.4 mg ofAcetyl CoA and 0.8U of purified enzyme. The concentrations of acetylatedproducts after ten minutes were determined by extraction from the TLCplates and measuring spectrophotometrically, in the case of Compound 6,and by counting radioactivity in the case of ¹⁴ C chloramphenicol. Thevelocity during the first ten minutes was assumed to be very close toV_(max). The velocity of Compound 6 was 0.66 of the velocity of ¹⁴ Cchloramphenicol.

EXAMPLE 51 Use of Compound 6 with Cellular Extracts

Cellular extracts from cultures of cerebral astrocytes from ratstransfected with the plasmid pENKAT-12 (CAT gene driven by theenkephalin promoter) were prepared. To 85 μL of this extract (in 0.25MTRIS pH 7.4) was added 33 μL TRIS pH 7.4, 0.5 mg Acetyl CoA and 7μL of a440 μM ethanol solution of Compound 6. The mixture was incubated at 37°C. for 4 hr. FIG. 1 shows the results of this experiment: lane 1 (fromthe left)--cells transfected, but not stimulated; lane 2--transfectedcells stimulated by 500μM 8-chlorophenylthio-cAMP; lane 3--transfectedcells stimulated by 10⁻⁶ M isoproterenol, a β-adrenergic stimulator;lane 4--positive control with purified CAT enzyme; lane 5--negativecontrol with no cell extract or enzyme.

EXAMPLE 52 Use of Fluorescent Compound in Assay of HIV-1

A specific and sensitive assay for functional AIDS virus (HIV-1) waspublished by Felber et al. This assay is based on cell lines containingthe CD4 receptor for HIV-1. The genome of these cell lines been alteredto contain the CAT gene under the control of the HIV regulatory region(long terminal repeat). Since HIV-1 virus produces its owntranscriptional activator, known as tat, which acts on the HIVregulatory region, presence of functional virus in the altered cell linewill induce transcription of not only the virus itself, but also the CATgene. If any material (including both cells and cell-free fluid)containing functional HIV-1 virus is introduced to a culture of thealtered cell line, CAT enzyme is induced greater than 500-fold overbasal levels. Therefore, one is able to detect actual HIV virus inclinical samples rather than antibodies to the virus whose productioncan be hindered by disease. This method could also be adapted to detectother viruses which produce specific activator proteins.

Proposed Procedure: Add the clinical sample to a dish containing thealtered cell line. Culture cells for 1 to 2 days. Either make a crudecell extract or incubate the cells with the fluorescent substrate.Measure the amount of acetylation of the substrate as described inExample 51. A significant amount of acetylation would be a specificindicator of actual HIV-1 virus in the clinical sample.

The use of a fluorescent CAT substrate rather than radiolabeledchloramphenicol would not only eliminate the problems of handling ofradioactive materials, but also speed the assay time (immediatedetection on a TLC plate versus sixteen hour autoradiography).Furthermore, fluorescence detection may allow a much simpler adaptationof this technique to automated analysis.

It is to be understood that, while the foregoing invention has beendescribed in detail by way of illustration and example, only thepreferred or specific embodiments have been revealed, and that numerousmodifications, substitutions, and alterations are all permissiblewithout departing from the spirit or scope of the invention as describedin the following claims.

We claim:
 1. A compound of the formula:

    BASE--N.sub.s --*X

a) where BASE is: ##STR4## substituted at one to five aromatic ringpositions by substituents, which may be the same or different, that arealkyl-, hydroxy-, alkoxy-, aryl-, halo-, nitro-, amino-, alkylamido-(where the alkyl portion contains 1-3 carbons, or arylamido- (where thearyl portion is a phenyl or alkyl-substituted phenyl) and n is greaterthan 0 and less than 6; and b) *X is a non-reduced tricyclicdifluoroboradiazaindacene fluorophore covalently bound to the terminalCH₂ of BASE through a linker N_(s) ; such that c) N_(s) --*X isamino-linked (--NH--*X), acetamido-linked (--NHCOCH₂ --*X),propionamido-linked (--NHCOCH₂ CH₂ --*X), sulfonamido-linked (--NHSO₂--*X), carboxamido-linked (--NHCO--*X), ureidyl-linked (--NHCONH--*X),thioureidyl-linked (--NHCSNH--*X), or linked by a covalent bond, to saidterminal CH₂ of BASE.
 2. A compound of the formula:

    BASE--N.sub.s --*X

a) where BASE is: ##STR5## where R is alkyl-, hydroxy-, alkoxy-, aryl-,halo-, nitro-, amino-, alkylamido- (where the alkyl portion contains 1-3carbons), or arylamido- (where the aryl portion is a phenyl oralkyl-substituted phenyl), and n is greater than 0 and less than 6; b)*X is a non-reduced tricyclic difluoroboradiazaindacene fluorophorecovalently bound to the terminal CH₂ of BASE through a linker N_(s) ;such that c) N_(s) --*X is amino-linked (--NH--*X), acetamido-linked(--NHCOCH₂ --*X), propionamido-linked (--NHCOCH₂ CH₂ --*X),sulfonamido-linked (--NHSO₂ --*X), carboxamido-linked (--NHCO--*X),ureidyl-linked (--NHCONH--*X), thioureidyl-linked (--NHCSNH--*X), orlinked by a covalent bond, to said terminal CH₂ of BASE.
 3. A compound,as claimed in claim 1, wherein the fluorophore *X has the formula:##STR6## where BASE-N_(s) -- is attached to said fluorophore at one ofS₁ -S₇, and the remainder of S₁ -S₇, which may be the same or different,are hydrogen or alkyl-.
 4. A compound, as claimed in claim 3, where BASEis substituted at one aromatic position by a substituent that isalkylamido-.
 5. A compound, as claimed in claim 4, where n=5 and N_(s)--*X is acetamido-linked (--NHCOCH₂ --*X) or propionamido-linked(--NHCOCH₂ CH₂ --*X) to said terminal CH₂ of BASE; or n is 1 or 2 andN_(s) is a covalent bond.
 6. A compound, as claimed in claim 2, whereinthe fluorophore *X has the formula: ##STR7## where BASE--N_(s) -- isattached to said fluorophore at one of S₁ -S₇, and the remainder of S₁-S₇, which may be the same or different, are hydrogen or alkyl-.
 7. Acompound, as claimed in claim 6, wherein R is acetamido-.
 8. A compound,as claimed in claim 7, wherein n is 5 and N_(s) is acetamido (--CH₂CONH--), or propionamido (--CH₂ CH₂ CONH--), or n is less than 3 N_(x)is a covalent bond.